Summary DNA interstrand cross-links (ICLs) covalently link the two strands of the DNA double helix and are extremely cytotoxic. Widely used chemotherapeutics (e.g. nitrogen mustards, cisplatin compounds) are thought to act through the generation of ICLs. However, tumors almost invariably become resistant to these agents, in part due to upregulation of DNA repair. ICLs are also generated by endogenous metabolites (e.g. reactive aldehydes, abasic sites), and failure to repair endogenous ICLs appears to cause human disease. For example, mutation in any one of 22 different FANC genes renders cells sensitive to ICLs and causes Fanconi anemia (FA), which is characterized by bone marrow failure and dramatically elevated predisposition to leukemia and other cancers. ICL repair by the FANC proteins occurs in S phase, but how they function was unknown. To elucidate mechanisms of ICL repair, we replicate plasmids containing site-specific ICLs in frog egg extracts. We showed previously that these extracts support cisplatin ICL repair by the FANC proteins. Repair requires that two replication forks converge on the ICL, which triggers the ubiquitylation and chromatin unloading of the replicative CMG helicase by the p97 ATPase. After CMG removal, the FANCI-FANCD2 complex stimulates dual incisions in the phosphodiester backbone on either side of the lesion, which resolves (unhooks) the ICL and creates a double-stranded DNA break (DSB) that is subsequently repaired by homologous recombination. In the last funding period, we discovered that the E3 ubiquitin ligase TRAIP is required for the ubiquitylation of CMG upon fork convergence. Interestingly, although TRAIP is always associated with replication forks, it only ubiquitylates CMGs upon fork convergence. In the last cycle, we also discovered a distinct mechanism of replication-coupled repair that operates on psoralen- and AP-ICLs. In this mechanism, CMG ubiquitylation by TRAIP leads to the recruitment of the NEIL3 DNA glycosylase, which cleaves one of the glycosyl bonds that form the ICL. Unhooking creates an abasic (AP) site in one strand but avoids formation of a DSB. In the current proposal, we use egg extracts and mammalian cells to address key questions that arise from our prior work. In Aim 1, we ask how CMG ubiquitylation by TRAIP is coupled to replication fork convergence, a critical feature of TRAIP function that avoids premature replisome disassembly. In Aim 2, we address how the NEIL3 pathway avoids formation of DSBs, the central feature that distinguishes it from the FA pathway. In Aim 3, we study the mechanism of an alternative response to ICLs called traverse, in which a single replication fork bypasses an intact ICL. The potential impact of these studies on human health is considerable because they will help create a solid foundation for the development of more effective cancer chemotherapies, as well as drugs that ameliorate the symptoms of Fanconi anemia.